The vascular permeability factor (PF) of Pseudomonas aeruginosa was produced by growing the organism in shallow cultures of a synthetic medium. The nucleic acids in the resultant viscous culture liquids were removed by the concomitant use of manganese chloride and sodium phosphate. After centrifugation, the supernatant was filtered through 450-nm membranes (Millipore) and concentrated with PM30 ultrafiltration membranes (Amicon). The concentrated material was further fractionated on Sephadex G-200 columns, which removed endotoxin and nucleic acids. The fractionated PF was then applied to DEAE-cellulose columns which had been equilibrated with 0.05 M Tris-HCl buffer, pH 7.6, and eluted with a linear gradient from 0.05 to 0.2 M NaCl. Analytical polyacrylamide gel electrophoresis revealed, however, that purified PF still contained several electrophoretically separable components. A considerable decrease in PF activity was noticed after Sephadex G-200 purification. However, PF activity was restored by the addition of a fraction which was excluded in the void volume of a column. Purified PF preparations exhibited cytopathic effect on various cell lines, and human embryonic lung cells and KB cells were found most sensitive. Unlike PF of Vibrio cholerae, PF of P. aeruginosa failed to produce fluid accumulation in ligated ileal loops of rabbits.